This invention relates to materials and methods for detecting nucleic acids in samples and, more particularly, to solid phase assays wherein transponders are associated with the beads constituting the solid phase, nucleic acid probes are bound to the surface of the particles and data concerning the assay is encoded on the transponders.
Solid phase assays have been used to determine the presence of nucleic acids, including deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and their modified forms. Solid-phase assays can be applied to nucleic acids either in simple buffers, or in biological fluids, such as blood, serum, plasma, saliva, urine, tissue homogenates, and many others.
In solid phase assays, small beads, or microparticles, are used as the solid phase to capture the analyte. Solid-phase microparticles can be made of different materials, such as glass, plastic, latex, depending on the particular application. Some beads are made of ferromagnetic materials to facilitate their separation from complex suspensions or mixtures.
In conventional solid-phase assays, the solid phase mainly aids in separating molecules that bind to the solid phase from molecules that do not bind to the solid phase. Separation can be facilitated by gravity, centrifugation, filtration, magnetism, immobilization of molecules onto the surface of the vessel, etc. The separation may be performed either in a single step in the assay or, more often, in multiple steps.
Often, there is a need to perform two or more different assays on the same sample, most of the time in a single vessel and at about the same time. Such assays are known in the art as multiplex assays. Multiplex assays are performed to determine simultaneously the presence or concentration of more than one molecule in the sample being analyzed, or alternatively, several characteristics of a single molecule, such as, the presence of several epitopes on a single protein molecule.
One problem with conventional multiplex assays is that they typically cannot detect more than about five analytes simultaneously, because of difficulties with simultaneous detection and differentiation of more than about five analytes. In other words, the number of different analytes that may be assayed in a single solid phase assay is limited by the solid phase. The present inventions overcomes these difficulties by providing a method of electronic indexing for different analytes.
High-throughput methods for genetic sequence analysis are critical for identification of polymorphisms and mutations in genes. Often, the first step in a genetic analysis is the DNA amplification using the polymerase chain reaction (PCR) to prepare a relatively large quantity of template (target) DNA. Ligase chain reaction (LCR) has also been used to prepare a quantity of the target DNA. The more genes and mutations in the gene one wants to investigate, the more amplification reactions one needs to perform. While in principle several amplification reaction can be performed in a single test tube, the identification and separation of the products of the amplification has presented a difficulty. The present invention provides the means of identifying products of the PCR and LCR reactions, as well as separating the products after the amplification reaction. The principle is the indexing of the oligonucleotides used for amplification in PCR or LCR by means of electronic circuitry.